Estrogen regulates gene transcription by binding to high affinity nuclear receptors (ERs), which then recruit co-activator proteins to the transcription complex. Two isoforms of ER (ER-atpha and ER-beta), with variable tissue distribution, have been described. Estrogen is thought to regulate the neuroendocrine reproductive axis by binding to ERs found in the hypothalamus and pituitary. To begin to understand estrogen regulation in vivo, targeted disruption of ER-alpha was performed. ER-alpha knock-out mice display significant gonadal defects and impaired feedback regulation of the neuroendocrine axis. However, after detailed study of this animal model, controversies still exist as to the effect of estrogen on the axis, the mechanism of estrogen's effect (direct versus indirect), and the level of regulation (hypothalamic, pituitary, or both). The goal of this project, therefore, is to determine the mechanism of estradiol action on the hypothalamus and pituitary using both in vitro and in vivo studies. This proposal has three specific aims. In Aim 1, the mechanism of estrogen regulation of the GnRtt neuron in vitro will be determined. ER mutants and GnRH reporter constructs will be tested in GnRH-expressing cell lines. Binding of ER complexes and cofactors to putative negative estrogen response sites in the GnRH promoter will also be evaluated. In Aim 2, the role of ER-alpha [or ER-beta] in the GnRH neuron versus pituitary gonadotroph will be studied in vivo. A conditional knockout of the ER-alpha [or ER-beta] locus in either the GnRH neuron or pituitary gonadotroph will be induced using Cre-LoxP technology. Finally in Aim 3, the mechanism of central estrogen feedback will be determined using cell-specific targeting of a mutant ERs to either the GnRH neuron and/or pituitary gonadotroph. The ability of these transgenes to rescue the defect observed in ER-alpha knock-out mice will also be tested. This proposal will provide an understanding of the molecular mechanism of estrogen feedback on the central reproductive axis.